Ni nta agarose and purification columns have the following specifications. The polyhistidine tag is the most popular affinity tag and typically consists of six consecutive histidine residues 6xhis. High resolution xray structure of tyvelose epimerase from. Nintaatto conjugates can be directly applied either to an sdspage gel or western blot membrane for fluorescence imaging, and have been successfully used in living cells.
Ninta agarose consists of the chelating ligand nitrilotriacetic acid nta immobilized on 6 % crosslinked agarose beads that are suitable for batch binding, gravity flow, and fplc columns. Ni nta agarose uses nta which represents the most commonly used chelating ligand in imac. If stored and handled correctly the beads have a 1 year shelf life. Purification of polyhistidinecontaining recombinant proteins. The nickelnitrilo triacetic acid ninta coupled to sepharose cl6b from qiagen offers a quick and efficient method of protein purification from small and large scale preparations obtained. Ni nta agarose beads are widely used for protein purification due to its high affinity and selectivity for recombinant fusion proteins that are tagged with six tandem histidine residues. Genscript antihis affinity resin is designed for purification of histagged protein.
Ni nta agarose 10 ml 10 ml disposable columns, 5 ml bedvolume 5 5 disposable columns, 1 ml bedvolume 5 5 frits for 5 ml 10 10 disposable columns. When using 96well blocks, cover block with tape and vortex 6 times for 5 s each time on a low to medium setting. Ninta column preparation resuspend ninta agarose slurry in a bottle container. Ninta is designed for high quality purification of 6xhistagged recombinant proteins from bacteria, insect and mammalian cells. The clarified lysate was loaded onto a 10ml ninta agarose qiagen inc. Add 10 ml ninta agarose slurry qiagen and incubate on rocker at 4c for 30min. How imidazole can be washed off the ninta agarose beads.
This resin can recover histagged proteins from a variety of expression systems such as baculovirus, yeast, mammalian and bacterial cells. Ni nta column preparation resuspend ni nta agarose slurry in a bottle container. Use of tandem affinity chromatography for purification of. Original manufacturer offers bulk volume of deae agarose, ni nta agarose. Histagged protein in the crude samples can bind to the antihis monoclonal antibodies coupled with the agarose, and the nonspecific p. Ninta magnetic agarose beads precharged with nickel captures 6xhistagged proteins, which can be used for studying proteinprotein i and dnaprotein interactions ii, and immunoassay procedures using antibodies specific for antigens present on the captured 6xhistagged biomolecule or antigens present on interaction partners bound to the.
Qiagen ni nta spin kit, 16 to 24m bead, up to 300gspin column binding capacity, cell lysate start material, automated processing, macroporus silica support, up to 95% homogeneity in one step, for fast, smallscale purification of 6xhis tagged proteins, ideal for proteomics application, includes 50 ni nta spin columns, reagents, buffers. All purification of recombinant proteins by ni nta chromatography for commercial purposes, and the commercial use of proteins so purified, require a license from hoffmannla roche. This information is helpful to other scientists as well as to. Can anybody help me to modify my method for ninta column. All chemicals are purchased from merck except for nintaagarose qiagen, glutathioneagarose sigma and coomassie brilliant blue g 250 serva. Xclose the column and mount the luer lock syringe without plunger as a buffer reservoir. Qiagen ninta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell. Ninta agarose purification of 6xhistagged proteins from e. Data file 1886 af affinity chromatography ni sepharose. Ninta superflow cartridge handbook 032007 7 introduction qiagen ninta superflow cartridges are prefilled with 1 ml or 5 ml ninta superflow and are ready to use for purification of 6xhistagged. For manual or automated purification of histagged proteins pdf 111kb. Dear all, i am using ninta agarose qiagen for purification of histagged proteins by gravityflow chromatography.
Pipet or pour 2 ml of the resin into a 10ml purification column supplied with the kit. Ni nta atto conjugates can be directly applied either to an sdspage gel or western blot membrane for fluorescence imaging, and have been successfully used in living cells. Pack a column with 1 ml ninta agarose and equilibrate it with 5 cv of buffer c containing 20 mm imidazole. Histidine residues in the his tag bind to the vacant positions in the coordination sphere of the immobilized nickel ions with high specificity and affinity. Pass the reaction mixture after tev cleavage though the small ni nta column by gravity to retain histagged products of cleavage as well as tev protease, and proceed with purification of the flowthrough. Ac501 purification histagged proteins nickel nta agarose. Nickel nta agarose beads are provided readytouse for rapid purification of histagged proteins under native or denaturing conditions.
These results are suggestive of similar binding affinity of mono and bisniiintamodified nanoparticles, probably a result of high surface coverage of. Over 20% more yield obtained with purecube ninta agarose. Ninta superflow cartridge handbook 032007 7 introduction qiagen ninta superflow cartridges are prefilled with 1 ml or 5 ml ninta superflow and are ready to use for purification of 6xhistagged proteins using a syringe, peristaltic pump, or liquid chromatography system such as the aktadesign or fplc system. Ni nta magnetic agarose beads handbook 122001 21 4. In vitro pulldown purified gstlrr3 or gst was mixed with hisralf22 in 1 ml buffer 20.
Ni affinity column can effectively purify extract even just one column. University of guelph biobar 2020 price list jan 1 through dec. Let the resin settle by gravity and gently aspirate the supernatant. All purification of recombinant proteins by ninta chromatography for commercial purposes, and the commercial use of proteins so purified, require a license from hoffmannla roche.
Add 10 ml ni nta agarose slurry qiagen and incubate on rocker at 4c for 30min. The clarified lysate was loaded onto a 10 ml ninta agarose qiagen inc. All reagents are guaranteed stable for 6 months when properly stored. Ni nta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Patents 4,683,195 and 4,683,202 and foreign equivalents owned by hoffmannla roche. Qiagen ninta spin kit, 16 to 24m bead, up to 300gspin column binding capacity, cell lysate start material, automated processing, macroporus silica support, up to 95% homogeneity in one step, for. Sonicate or homogenize on ice to lyse cells 6 times for 10 s each time with 5 s pauses between. Ninta magnetic agarose beads precharged with nickel captures 6xhistagged proteins, which can be used for studying proteinprotein i and dnaprotein. Xequilibrate the column with 10 to 15 bed vo lumes 6 9 ml of equilibration buffer. It is intended for use in gravity or pressure flow columns, and batch purifications. Ninta lysis buffer containing 50 mm nah 2 po 4, 300 mm nacl, and 10 mm imidazole at ph 8.
Highlyefficient purification of native polyhistidinetagged. Basel, switzerland, who have purified more than 100 different proteins on ni nta resin, we recommend a maximum of 5 runs per column. Qiagen biobar university of guelph pricelist valid from sept. Ninta agarose is an affinity chromatography matrix for purifying recombinant proteins carrying a his tag. Reuse of ninta resin the reuse of ninta resin depends on the nature of the sample and should only be performed with identical recombinant proteins. Histidine residues in the his tag bind to the vacant positions in the coordination sphere of the.
Pack a column with 1 ml ni nta agarose and equilibrate it with 5 cv of buffer c containing 20 mm imidazole. Ni nta agarose consists of the chelating ligand nitrilotriacetic acid nta immobilized on 6 % crosslinked agarose beads that are suitable for batch binding, gravity flow, and fplc columns. No secondary reaction is required, since the ni nta complex is. This resin consists of crosslinked agarose derivatized with nitrilotriacetic acid nta and provides good properties working in native or denaturing conditions. Ninta purification system thermo fisher scientific. Jul 28, 2003 all chemicals are purchased from merck except for nintaagarose qiagen, glutathioneagarose sigma and coomassie brilliant blue g 250 serva.
It is intended for use in gravity or pressure flow columns, and batch. Cgi114 protein concentrations were measured by absorbance at 280 nm mw 26,275. Ni nta agarose, ni nta superflow manufacturersupplier. Ni nta magnetic agarose beads are used for smallscale affinity purification as well as highthroughput screening of recombinant histagged proteins. Xfill the column with ninta resin to create a bed volume of 0. Qiagen ninta agarose, 100ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start. Ni affinity column can effectively purify extract even just one.
Allow the resin to settle completely by gravity 510 minutes or gently pellet it by lowspeed centrifugation 1 minute at 800. Given that this agarose is remarkably expensive i would like to reuse it. This step may need to be optimized for different cell cultures and vortexers. For purification of histagged proteins by gravityflow chromatography. Oem manufacturer in china, for ni nta agarose, deae agarose.
Cellular debris was removed by centrifugation at 4 c for 25 min at 20,000 g. After loading, the column was washed with about 60 mls of ninta wash buffer. The polyhistidine tag is the most popular affinity tag. Cells were lysed on ice by six cycles of sonication 30 s separated by 2 min of cooling. Qiagen customers are also a major source of information regarding advanced or specialized uses of our products. Qiagen ninta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start. The his 6tag was removed by tev protease treatment at 4c for 16 hr. Nebexpress ni resin is an affinity matrix for the isolation and purification of polyhistidinetagged histagged fusion proteins. Ninta technology 18 ninta chromatographic materials 19 cloning 21 choosing a qiaexpress construct 21 intended use of recombinant proteins and pqe vector choice 22 protein size 22 codon. Ninta agarose and purification columns have the following specifications. Storage conditions the beads are shipped at ambient temperature.
Below, nickel leakage and other performance data for ni sepharose 6 fast flow have been compared with other products on the market also intended for histidinetagged protein purificationninta. It is composed of agarose and conjugated thetm antihis monoclonal antibody. Ni nta is designed for high quality purification of 6xhistagged recombinant proteins from bacteria, insect and mammalian cells. Dear all, i am using ni nta agarose qiagen for purification of histagged proteins by gravityflow chromatography. Reuse of ni nta resin the reuse of ni nta resin depends on the nature of the sample and should only be performed with identical recombinant proteins. Sep 06, 2007 the nickelnitrilo triacetic acid ni nta coupled to sepharose cl6b from qiagen offers a quick and efficient method of protein purification from small and large scale preparations obtained through variable expression sources like e. Glutathione sepharose 4b ge healthcare, and hisralf22 was purified by using ninta agarose qiagen.
Ninta magnetic agarose beads are used for smallscale affinity purification as well as highthroughput screening of recombinant histagged proteins. Invitrogen novex ninta purification system 6 purifications. Add 6 ml of sterile distilled water and resuspend resin. Ni nta technology 18 ni nta chromatographic materials 19 cloning 21 choosing a qiaexpress construct 21 intended use of recombinant proteins and pqe vector choice 22 protein size 22 codon usage 23 internal start sites 23 inefficient translation 23 secretion 23 cloning procedures and vector maps 24 procedure for direct cloning of pcr fragments. Qiagen ni nta agarose, 25ml, 45 to 165m bead, up to 50mgml binding capacity, cell lysate start material, nickelcharged resin, 2. Ni nta agarose, deae agarose, deae cellulose de52, cm.
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